Oberhammer F, Fritsch G, Schmied M, Pavelka M, Printz D, Purchio T, Lassmann H, Schulte-Hermann R
Institute of Tumorbiology-Cancer Research, Department of Toxicology, University of Vienna, Austria.
J Cell Sci. 1993 Feb;104 ( Pt 2):317-26. doi: 10.1242/jcs.104.2.317.
A current hypothesis holds that chromatin fragmentation into oligonucleosomal patterns is an early event during apoptosis. In contrast, induction of apoptosis in cultured hepatocytes by TGF-beta 1 was not associated with DNA fragmentation into oligonucleosomes in hepatocyte monolayers and apoptotic fragments. For a more rigorous test of the hypothesis we performed a number of experiments. We compared nuclear changes resulting from TGF-beta 1 with those induced by Ca2+, a known activator of endonuclease. The morphology of apoptotic and Ca(2+)-treated nuclei was different as judged by DNA staining with Hoechst 33258. Likewise, electron microscopy of apoptotic nuclei showed characteristic condensation of the chromatin as well as dissolution of the nucleolar structure and nuclear fragmentation, changes not seen after Ca2+ treatment, after three hours of incubation. Analysis of DNA fluorescence of nuclei by FACS revealed that treatment with Ca2+ reduced the signal by 20%. In contrast, nuclei from TGF-beta 1-treated hepatocytes did not exhibit a reduced signal and after sorting by FACS, apoptotic nuclei remained in the 2N and 4N fractions. The absence of detectable DNA fragmentation in apoptotic nuclei was further verified by in situ nick translation, not only in hepatocytes but also in a mouse lymphoma cell line. From these findings we conclude that activation of an endonuclease is not an early event on the pathway to morphologically recognizable apoptosis.
目前的一种假说认为，染色质断裂成寡核小体模式是细胞凋亡过程中的早期事件。相比之下，转化生长因子β1（TGF-β1）诱导培养的肝细胞凋亡时，在肝细胞单层和凋亡碎片中，DNA并未断裂成寡核小体。为了更严格地验证这一假说，我们进行了一系列实验。我们将TGF-β1引起的核变化与已知的核酸内切酶激活剂Ca2+诱导的核变化进行了比较。用Hoechst 33258对DNA进行染色判断，凋亡细胞核和经Ca2+处理的细胞核形态不同。同样，凋亡细胞核的电子显微镜检查显示染色质有特征性的凝聚以及核仁结构的溶解和核碎片化，而在孵育三小时后，经Ca2+处理的细胞核未见这些变化。通过流式细胞术（FACS）分析细胞核的DNA荧光发现，Ca2+处理使信号降低了20%。相比之下，TGF-β1处理的肝细胞的细胞核未显示信号降低，并且经FACS分选后，凋亡细胞核仍留在2N和4N组分中。原位缺口平移进一步证实了凋亡细胞核中未检测到DNA断裂，不仅在肝细胞中如此，在小鼠淋巴瘤细胞系中也是如此。从这些发现我们得出结论，核酸内切酶的激活不是形态学上可识别的细胞凋亡途径中的早期事件。
